This invention relates to certain crystallized kinase protein-ligand complexes, particularly complexes of crystallized P38 protein, and more particularly complexes of P38xcex3 protein. This invention also relates to crystallizable compositions from which the protein-ligand complexes may be obtained. This invention also relates to computational methods of using structure coordinates of the protein complex to screen for and design compounds that interact with the protein, particularly P38 protein or homologues thereof.
Mammalian cells respond to extracellular stimuli by activating signaling cascades that are mediated by members of the mitogen-activated protein (MAP) kinase family. Mammalian mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that facilitate signal translocation in cells [Davis, Mol. Reprod. Dev. 42, 459-467 (1995); Cobb et al., J. Biol. Chem. 270, 14843-14846 (1995); Marshall, Cell 80, 179-185 (1995)]. MAP kinases include the extracellular-signal regulated kinases (ERKs), the c-Jun NH2-terminal kinases (JNKs) and the P38 kinases, which have similar sequences and three-dimensional structures [Taylor and lipopolysaccharides (LPS), UV, anisomycin, or osmotic shock, and by cytokines, such as interleukin-1 (IL-1) and tissue necrosis factor (TNF). Inhibition of P38xcex1 kinase leads to a blockade on the production of both IL-1 and TNF. IL-1 and TNF stimulate the production of other proinflammatory cytokines such as IL-6 and IL-8 and have been implicated in acute and chronic inflammatory diseases and in post-menopausal osteoporosis [Kimble et al., Endocrinol., 136, 3054-61 (1995)].
Based upon this finding it is believed that P38xcex1, along with other MAPKs, has a role in mediating cellular response to inflammatory stimuli, such as leukocyte accumulation, macrophage/monocyte activation, tissue resorption, fever, acute phase responses and neutrophilia. In addition, the MAPKs, such as P38xcex1, have been implicated in cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and neurodegenerative disorders. Inhibitors of P38xcex1 also appear to be involved in pain management through inhibition of prostaglandin endoperoxide synthase-2 induction. Other diseases associated with Il-1, IL-6, IL-8 or TNF overproduction are set forth in WO 96/21654. P38xcex3 MAP kinase (also known as ERK6 and stress activated protein kinase-3 or SAPK3) is a newly discovered member of the MAP kinase family. However, unlike the other P38 family members which are expressed in many tissues, P38xcex3 is expressed at highest levels in skeletal muscle [Li et al., Biochem Biophys Res Commun 228, 334-340 (1996); Enslen et al., J Biol Chem 273, 1741-1748 (1998); Raingeaud et al., J. Biol. Chem. 270, 7420-7426 (1995)]. Thus P38xcex3 may have a unique function related to muscle morphogenesis, and it may be a potential target for treating degenerative diseases occurring in muscle tissue.
Compounds that selectively inhibit P38xcex3 and not P38xcex1 would be highly desirable. It would be useful to have new treatments for muscle degenerative diseases using compounds that do not suppress the inflammatory response or other functions of P38xcex1. However, the design of inhibitors that are selective for any particular MAP kinase, such as P38xcex3, is challenging due to the structural similarity of the MAP kinases. Therefore, it would be advantageous to have a detailed understanding of the structures of the various MAP kinases in order to exploit any subtle differences that may exist among them.
A general approach to designing inhibitors that are selective for an enzyme target is to determine how a putative inhibitor interacts with the three dimensional structure of the enzyme. For this reason it is useful to obtain the enzyme protein in crystal form and perform X-ray diffraction techniques to determine its three dimensional structure coordinates. If the enzyme is crystallized as a complex with a ligand, one can determine both the shape of the enzyme binding pocket when bound to the ligand, as well as the amino acid residues that are capable of close contact with the ligand. By knowing the shape and amino acid residues in the binding pocket, one may design new ligands that will interact favorably with the enzyme. With such structural information, available computational methods may be used to predict how strong the ligand binding interaction will be. Such methods thus enable the design of inhibitors that bind strongly, as well as selectively to the target enzyme.
Crystal structures are known for some of the MAP kinases; for example, unphosphorylated JNK3, unphosphorylated P38xcex1, and ERK2 in both phosphorylated and unphosphorylated forms. Phosphorylated ERK2 is reported to exist as a dimer in both solution and as a crystal. The unphosphorylated forms of JNK3, ERK2 and P38xcex1, on the other hand, are reported to be monomeric. [Tong et al., Nat Struct Biol 4, 311-316 (1997); Wilson and Su, Chem Biol 4, 423-431 (1997); Xie et al., Structure 6, 983-991 (1998); Zhang et al., Nature 367, 704-711 (1994); Canagarajah et al., Cell 90, 859-869 (1997); Wilson and Su, J Biol Chem 271, 27696-27700 (1996)].
The crystal structure reported for P38xcex1 is based on unphosphorylated protein. However, it is the phosphorylated or activated form of the enzyme that is able to phosphorylate its substrate enzyme. In order to disrupt the phosphorylation of the substrate, and produce the desired clinical effect, a small molecule inhibitor would likely act by blocking a phosphorylated form of P38. Thus, the most suitable target for drug design is the active or phosphorylated form. While the structure of the unphosphorylated enzyme is often used for drug design purposes, there is an inherent uncertainty as to whether the phosphorylated and unphosphorylated forms would bind a designed inhibitor with equal affinity.
A class of pyridinylimidazole compounds are known to inhibit P38xcex1 MAP kinase [Lee et al., Nature 372, 739-746 (1994)]. These inhibitors have been shown to bind in the ATP binding site of P38xcex1 [Young et al., J Biol Chem 272, 12116-12121 (1997); Tong et al., Nat Struct Biol 4, 311-316 (1997); Wilson et al., Chem Biol 4, 423-431 (1997)]. However, the pyridinylimidazoles reportedly do not inhibit the activity of ERK2, JNK3, or P38xcex3. This observed selectivity is interesting because the amino acid sequence in the ATP binding site of the various kinases are known to be highly conserved [Fox et al., Protein Science 7, 2249-2255 (1998); Xie et al., supra; Wilson and Su, supra; Enslen et al., J Biol Chem 273, 1741-1748 (1998)].
As there is a need for compounds that selectively inhibit a particular MAP kinase, it would be desirable to have improved methods that facilitate the design of such compounds. For this purpose, knowledge of the three dimensional structure coordinates of an activated P38 protein would be useful. Such information would aid in identifying and designing potential inhibitors of particular P38 proteins which, in turn, are expected to have therapeutic utility.
This invention provides certain crystallized, protein kinase-ligand complexes, in particular P38-ligand complexes, and their structure coordinates. The structure coordinates are based on the structure of a phosphorylated P38xcex3-ligand complex that has now been solved and which reveals new structural information useful for understanding the activated states of other, related kinase proteins as described herein. The key structural features of the proteins, particularly the shape of the substrate binding site, are useful in methods for designing or identifying selective inhibitors of the protein kinases, particularly P38, and in solving the structures of other proteins with similar features.
The invention also provides a computer which which is programmed with the structure coordinates of the activated P38 binding site. Such a computer, appropriately programmed and attached to the necessary viewing device, is capable of displaying a three-dimensional graphical representation of a molecule or molecular complex comprising such binding sites or similarly shaped homologous binding pockets.
The invention also provides a method for determining at least a portion of the three-dimensional structure of other molecules or molecular complexes which contain at least some features that are structurally similar to P38xcex3, particularly P38xcex1, P38xcex2, P38xcex4 and other P38 isoforms. This is achieved by using at least some of the structural coordinates obtained for a phosphorylated P38 complex.